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Abstract

Clinical observation suggest that bone formation is influenced by the environmental
niche where it takes place and specifically that soft connective tissues may inhibit the
bone healing process. The aim of the studies described in this thesis is to test the
hypothesis that fibroblasts inhibit the differentiation and function of osteoblasts in vitro.
To address this aim the ability of fibroblasts and their supernatants to inhibit osteoblast
differentiation was investigated. In addition, the inhibitory effects of gingival and
periodontal ligament fibroblasts were compared. Next Bone Morphogenetic Proteins
(BMPs) and their antagonists were tested for their ability to modulate the activity of
fibroblast supernatants. Finally the effects of fibroblast supernatants on osteoblast
chemotactic responses were investigated.
Primary fibroblasts were isolated from rat gingivae, skin, oral mucosa and periodontal
ligament (PDL) using explant cultures. Primary osteoblast cultures were established by
enzymatic digestion of neonatal rat calvariae. In other experiments the osteoblastic cell
line ROS 17/2.8 was used. Osteoblast differentiation was assessed by measuring
alkaline phosphatase (ALP).
In co-culture experiments using 3-D collagen gels and diffusion chamber inserts
fibroblasts strongly inhibited osteoblast differentiation. Furthermore conditioned medium
from superficial connective tissues fibroblasts consistently inhibited osteoblast
differentiation (greater than 50% inhibition of ALP expression. In contrast, PDL cells
strongly stimulated ALP expression (greater than 100% increase). Stimulation of ROS
17/2.8 cells with BMP-2 increased ALP expression (more than 3 fold increase with
10ng/ml BMP-2), and this effect could be completely blocked by the BMP-antagonist
100ng/ml noggin. Similarly conditioned media from gingival, oral mucosal and skin
fibroblasts totally suppressed the effects of BMP-2. In contrast, PDL conditioned media
stimulated ALP expression in additively with BMP-2, and his effect could also be
blocked by noggin.
Using a micro-well Boyden Chamber both PDGF and BMP-2 caused a dose-dependent
increase in chemotaxis. However fibroblast conditioned medium totally blocked the
chemotactic effects on BMP-2, but had no effect on PDGF-induced chemotaxis.
Overall these studies demonstrate that fibroblasts from superficial connective tissues
(gingival, oral mucosa and skin) can inhibit osteoblast function by secretion of BMPantagonists
and that superficial connective tissues and PDL are distinct in respect to
their role in bone healing. Further studies are needed to identify the specific molecular
identity of this inhibitory activity and to extend these observations to an in vitro model.
However in the longer term it is proposed that information on the regional expression of
BMP inhibitors may lead to novel therapeutic interventions to promote bone growth in
periodontal and implant related bone regeneration procedures.